Palladin is a novel 92 kD protein that appears to play a critical role in the assembly of actin-based structural arrays. We hypothesize that palladin is a key scaffold protein that provides docking sites for multiple proteins that regulate the assembly and higher-order organization of actin filaments. Since the actin cytoskeleton provides the structural framework for many fundamental cellular processes (including spreading, adhesion, and migration), it is important to understand exactly how palladin functions to modulate cytoskeletal organization. To this end, the following questions will be addressed: (1) What are the structural and regulatory proteins of the cytoskeleton that bind to palladin? For these studies, we will be guided by sequence homologies that are shared between palladin and known cytoskeletal proteins, to direct us to potential binding partners. (2) How is palladin regulated? To answer this question, we will take advantage of the observation that palladin becomes modified when cells round up to enter mitosis. We will investigate the biochemical nature of this modification, and we will explore how mitosis-specific modifications of palladin impact upon its ability to bind to its usual partners. (3) What domains of palladin are required for its binding interactions? Docking sites on palladin will be mapped to specific amino acids, so that we can design palladin mutants that are deficient in the ability to provide certain binding sites. These palladin mutants will then be expressed in living cells, to explore the effects on cytoskeletal assembly. In summary, we believe that palladin is a unique and powerful tool with which to study the cellular mechanisms that govern how the cytoskeleton is assembled, maintained and regulated in living cells. Cell spreading, adhesion and migration are critical for processes such as wound healing, inflammation reaction, neurite outgrowth and cancer metastasis; thus, a better understanding of the regulation of actin-based structures could have potential health benefits in many different fields.